The general aim of this project is to study the effects of immunotoxin induced transient T-cell depletion on the modulation of T cells from immunization responses towards tolerizing responses, and to use this knowledge for the experimental and clinical treatment of T cell driven autoimmune diseases, graft-versus-host disease, and the induction of tolerance to mismatched organ and cell transplants. We previously showed that a 2-3 day course of anti-rhesus CD3 immunotoxin constructed with CRM9, a binding site mutant of diphtheria toxin, depletes lymph node and blood T cells by 99% after a 48 hour lag period. This process was associated with a marked prolongation of survival of mismatched functioning rhesus kidney transplants, 50% over 9 months without further immunosuppressive therapy. Rejections, were preceded by the appearance of anti-graft antibodies (done with S. Knechtle and J. Thomas). To circumvent this adverse T cell mediated B cell response that is apparently initiated during the delay in immunotoxin induced T cell killing, we have added a short course of agents that are believed to block antigen presentation such as deoxyspergualin (DSG). Out of 5 bilaterally rephrectomized recipients long term tolerance was achieved in 3 without further immunosuppresion as judged by a graft survival of 4 years or longer. Congeneic pancreatic islet cell transplants in monkeys suffering from spontaneous insulin dependent diabetes have been shown to reverse diabetes as judged by returning non-fasting blood glucose and glycosylated hemoglobin values to normal in the absence of exogenous insulin. A short course of immunotoxin, methyl prednisolone and cyclosporine provide induction of stable operational tolerance to islets that has lasted over one year in one case. This is the first reported reversal of diabetes using a tolerizing protocol that does not depend on chronic immunosuppression, a process previously shown to compromise transplanted islet function (done with J. Thomas). Streptozotocin induced diabetes in monkeys has been successfully reversed by using anti-T cell immunotoxin in combination with 14 days of DSG and allografted pancreatic islets (done with J. Thomas). Three out of seven are long term tolerant survivors (6-7 years). A recombinant divalent anti-human anti-T cell immunotoxin has been developed that has 20-fold the potency of the previous chemical conjugates. This is expressed in Pichia pastoris. This material is now under production for a phase I clinical trial in T cell leukemia and aplastic anemia, a T cell driven autoimmune disease. In order to aid the production of immunotoxin and other bioactive proteins in Pichia pastoris, new strains have been constructed that exhibit toxin resistance and increased heterologous protein production in the presence of an active unfolded-protein response.